Advances in Lignocellulosic Biomass Hydrolyzing Enzymes
ID: 3413

Abstract: Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century.

For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this panel, we will review the advances in cellulase research and developments and focus on structure and function. Cellulase improvement strategies based on

directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using

insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library, and advances in anaerobic fungal cellulases research.



Anu Koivula

Cellulases are important enzymes for industrial applications and have proved commercially useful e.g. in textile, detergent, and pulp and paper industry. Cellulase research has recently focussed on the conversion of lignocellulosic biomass to fermentable sugars as an alternative to fossil fuels. Commercially available cellulases are often derived from mesophilic micro-organisms such as Trichoderma reesei and the hydrolysis process is performed at 40-50 °C due to instability of these enzymes at higher temperatures. Thermostable enzymes would provide improved process options making use of the elevated temperatures. The general robustness and better overall stability of the thermophilic enzymes compared to the mesophilic ones could also increase the inhibitor tolerance and recyclability of the enzymes.

Glycosyl hydrolase family 7 (GH-7; see http://afmb.cnrs-mrs.fr/CAZY/citing.html) cellobiohydrolases seem to be especially important in the hydrolysis of highly crystalline cellulose, and are found only in the fungal kingdom. Here, we present the characterisation of the enzymatic properties of three novel thermostable cellobiohydrolases originating from the thermophililic fungi Acremonium thermophilum, Chaetomium thermophilum and Thermoascus aurantiacus. These GH-7 family enzymes were expressed in the industrially relevant production host T. reesei, and the kinetics on small soluble substrates, cellobiose inhibition, crystalline cellulose hydrolysis and thermostability of the purified enzymes were determined. Enzymatic properties of the cellobiohydrolases were compared to those of T.reesei cellobiohydrolase Cel7A, one of the most thoroughly studied fungal cellobiohydrolases. The best Cel7 cellobiohydrolase was also tested in hydrolysis of pretreated materials.

Acknowledgements: This work was supported by a grant from the EU (“Technological improvement for ethanol production from lignocellulose” project; coordinator L. Viikari, VTT).

References:

[1] Voutilainen, S., Puranen, T., Siika-aho, M., Lappalainen, A., Alapuranen, M., Kallio, J., Hooman, S., Viikari, L., Vehmaanperä, J. and Koivula, A. (2008) Cloning, expression and characterization of novel thermostable family 7 cellobiohydrolases. Biotechn. Bioeng.101, 515-528.

[2] Öhgren K, Vehmaanperä J, Siika-Aho M, Galbe M, Viikari L, Zacchi G. (2007) High temperature enzymatic prehydrolysis prior to simultaneous saccharification and fermentation of steam pretreated corn stover for ethanol production. Enzyme Microb Technol 40, 607-613.

[3] Viikari, L., Alapuranen,M., Puranen, T., Vehmaanperä, J. and Siika-aho, M. (2007) Thermostable Enzymes in Lignocellulose Hydrolysis. Adv Biochem Engin/Biotechnol, 108: 121–145.



Masahiro Samejima

In order to introduce enzymatic saccharification into the production of ethanol fuels from cellulosic biomasses, a reduction in the dosage of cellulase is required through dramatic improvement in enzymatic activity for crystalline celluloses. Efficient enzymatic saccharification of crystalline cellulose (10-times higher than that with conventional condition) has been achieved by conversion of crystalline substrate from cellulose I to cellulose III with ammonia treatment. This improvement is caused by significant increase in the productive binding of cellulase on the surface of cellulose. Moreover, by selection of an appropriate cellulase for the new substrate, 40-time higher efficiency has been attained compared with conventional condition. These observations clearly suggest us that binding control of cellulase on cellulosic biomass must be an important way towards technology innovation for the target.



Manoj Kumar

DSM is developing a commercially viable enzymes system for lignocellulosic biomass saccahrification using a differentiable and key competence driven fungal production base of enzymes. DSM has a competitive LC biomass saccharification enzyme system that is currently cost competitive to the other leading developmental products. DSM is a leading global commercial enzyme supplier and enzyme research and development powerhouse that traces its enzyme biotechnology roots from Gist Brocades and Roche. DSM possesses extensive fungal expression system knowhow, has an internal collection of commercially fermentable fungal hosts, possesses genomic and secretomic information of various fungal hosts for enzyme expression and commercial production such as xylanases, proteases, lipases, carbohydrases, and oxidases.

DSM will describe latest results on LC biomass hydrolyzing enzymes system that incorporates pretreated biomass feed-stock based fungal fermentation to optimize various cellulolytic enzyme cascade system.



Steven Hutcheson

Bacteria and fungi are thought to degrade cellulose to cellobiose through the activity of either a complexed or noncomplexed cellulolytic system composed of endoglucanases and cellobiohydrolases. The marine bacterium Saccharophagus degradans 2-40 produces a novel multi-component cellulolytic system that lacks a cellobiohydrolase but has an abundance of annotated endoglucanases. Activities for each component of the cellulolytic system have been established. Three of the endoglucanases showed significantly higher activity on several types of cellulose and processively-released cellobiose from cellulosic substrates. The processive-endoglucanases were found to be functionally equivalent to the endoglucanases & cellobiohydrolases required for other cellulolytic systems, thus providing an efficient mechanism for this bacterium to convert cellulose to cellobiose.





Moderator: Manoj Kumar, DSM, NV (United States)

Presenter 1: Improvement of Cellulase and Cellulose Interaction Towards Technology Innovation On Biofuel Production From Cellulosic Biomass
MASAHIRO SAMEJIMA, The University of Tokyo, (Japan)  [Confirmed]

Presenter 2: Novel-Thermostable Family 7 (GH-7) Cellbiohydrates For Cellulose Hydrolysis 
ANU KOIVULA, VTT Technical Research Centre of Finland, (Finland)  [Confirmed]

Presenter 3: DSM Enzymes System for Biomass Saccharification 
Manoj Kumar, DSM, NV, (United States)  [Confirmed]

Presenter 4 (if necessary): Novelties of A Bacterial Cellulolytic System Applicable To Cellulosic Sugar Production 
Steven Hutcheson, Zymetis, Inc, (United States)  [Confirmed]

Panel Organizer:
Manoj Kumar, DSM, NV, (United States)

Why should your submission should be selected for this year’s program?
THIS PANEL WILL PROVIDE NEW DEVELOPMENTS AND ADVANCES MADE IN CELLULASE KNOWLEDGE THAT WILL HELP AND PROVIDE ATTENDEES NEW INSIGHTS AND IDEAS ON HOW TO IMPROVE CELLULASES FOR THEIR EFFICIENCY AND COST FOR BIOMASS SACCHARIFICATION. PANEL INCLUDES, WELL KNOWN INSTITUTIONS ACTIVE AT THE FOREFRONT OF BIOMASS ENZYME RESEARCH